Lysin agent and method of use for diagnostic testing

Abstract

Disclosed herein are the identification, cloning, and optimizing the lytic activity of one or more novel Bacillus lysin proteins, a method of selecting a lysin agent for use in molecular diagnostic testing that includes analyzing genome databases for Bacillus anthracis and near neighbors, selecting candidate genes encoding potential lytic enzymes based on conserved amino acid motifs as determined in peptidoglycan hydrolases, cloning the candidate genes in expression vector, isolating proteins thereof, and testing for lytic activity against Bacillus anthracis, and selecting an optimum gene of the candidate genes for optimizing lysis conditions.

What is claimed: 
     
       1. A recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ. ID NO: 2, or fusion thereof. 
     
     
       2. The recombinant nucleic acid of  claim 1 , wherein the nucleotide sequence is operably linked to one or more control sequences. 
     
     
       3. The recombinant nucleic acid of  claim 2 , wherein the one or more control sequences is selected from a group consisting of: a promoter, a transcriptional start signal, a transcriptional stop signal, a translational start signal, and a translational stop signal. 
     
     
       4. The recombinant nucleic acid of  claim 1 , wherein the nucleotide sequence comprises SEQ ID NO: 1. 
     
     
       5. A vector comprising a recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a fusion thereof. 
     
     
       6. The vector of  claim 5 , wherein the nucleotide sequence is operably linked to one or more control sequences. 
     
     
       7. The vector of  claim 6 , wherein the one or more control sequences is selected from a group consisting of: a promoter, a transcriptional start signal, a transcriptional stop signal, a translational start signal, and a translational stop signal. 
     
     
       8. The vector of  claim 5 , wherein the nucleotide sequence comprises SEQ ID NO: 1. 
     
     
       9. A host cell transformed with a vector comprising a recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the am no acid sequence of SEQ ID NO: 2, or a fusion thereof. 
     
     
       10. The host cell of  claim 9 , wherein the nucleotide sequence is operably linked to one or more control sequences. 
     
     
       11. The host cell of  claim 10 , wherein the one or more control sequences is selected from a group consisting of: a promoter, a transcriptional start signal, a transcriptional stop signal, a translational start signal, and a translational stop signal. 
     
     
       12. The host cell of  claim 9 , wherein the nucleotide sequence comprises SEQ ID NO: 1. 
     
     
       13. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a fusion thereof, the polypeptide having peptidoglycan hydrolase activity. 
     
     
       14. A method for producing a polypeptide with peptidoglycan hydrolase activity, comprising:
 (a) providing a host cell comprising a vector comprising a nucleotide sequence encoding the polypeptide with peptidoglycan hydrolase activity, wherein the nucleotide sequence is operably linked to one or more control sequences; and 
 (b) culturing the host cell under culturing conditions; and 
 (c) isolating the polypeptide, 
 wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. 
 
     
     
       15. The method of  claim 14 , wherein the one or more control sequences is selected from a group consisting of: a promoter, a transcriptional start signal, a transcriptional stop signal, a translational start signal, and a translational stop signal. 
     
     
       16. The method of  claim 14 , wherein the nucleotide sequence comprises SEQ ID NO: 1. 
     
     
       17. A method for diagnosing the presence or absence of bacteria in a sample comprising:
 (a) providing a sample of the bacteria; and 
 (b) incubating the sample of the bacteria in the presence of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a fusion thereof, the polypeptide having peptidoglycan hydrolase activity, and 
 (c) contacting the sample and the isolated polypeptide having peptidoglycan hydrolase activity with at least a first and a second oligonucleotide primer under conditions sufficient to provide polymerase-based nucleic acid amplification; and 
 (d) detecting the presence or absence of an amplification product. 
 
     
     
       18. The method of  claim 17 , wherein the bacteria is a gram-positive bacteria. 
     
     
       19. The method of  claim 18 , wherein the gram-positive bacteria is a  Bacillus  species. 
     
     
       20. The method of  claim 19 , wherein the  Bacillus  species is  Bacillus anthracis.    
     
     
       21. The method of  claim 17 , wherein the bacteria is a gram-negative bacteria. 
     
     
       22. The method of  claim 21 , wherein the gram-negative bacteria is a  Yersinia  species. 
     
     
       23. The method of  claim 22 , wherein the  Yersinia  species is  Yersinia pestis.    
     
     
       24. The method of  claim 17 , wherein the polymerase-based nucleic acid amplification is quantitative polymerase chain reaction (QPCR). 
     
     
       25. A method for lysing bacteria comprising:
 (a) providing a sample of the bacteria; and 
 (b) preparing an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a fusion thereof, the polypeptide having peptidoglycan hydrolase activity; and 
 (c) incubating the isolated polypeptide with the bacteria. 
 
     
     
       26. The method of  claim 25 , wherein the bacteria is a gram-positive bacteria. 
     
     
       27. The method of  claim 26 , wherein the gram-positive bacteria is a  Bacillus  species. 
     
     
       28. The method of  claim 27 , wherein the  Bacillus  species is  Bacillus anthracis.    
     
     
       29. The method of  claim 25 , wherein the bacteria is a gram-negative bacteria. 
     
     
       30. The method of  claim 29 , wherein the gram-negative bacteria is a  Yersinia  species. 
     
     
       31. The method of  claim 30 , wherein the  Yersinia  species is  Yersinia pestis.